| Objective:In this study, we prepared the experimental micromovement model of rabbit femur fracture using self-designed mini-external-fixator. and realize the size and formation of callus through dynamic observation of X-ray’s performance on different time point, and detected the BMD and callus mechanical characteristics, and analyzed the histology and immunohistochemistry results in order to tried to explore the influence and mechanism of time effect of the controlled micromovement on fracture healing.Methods:we prepared48experimental micromovement model of rabbit femur fracture, which were divided randomly into four groups(with12rabbits for each group), all were fixed with unilateral external fixator connected with two bars. Group one:continuing immobilization group, There exists no micromovement between the fragment after the fracture model was made and fixed with unilateral two bars external fixator. Group two:instant micromovement group, the0.5mm micromovement was induced immediately by the sliding between the two connection bars. Group three:micromovement began after1week, the0.5mm micromovement was induced after1week’s immobilization. Group four:micromovement began after2week, the0.5mm micromovement was induced after2week’s immobilization. The AP and lateral view of the femur were obtained at1、2、3and5weeks postoperatively, and observe the callus formation according to the score of the external callus formation and the vagueness level of fracture line. After5weeks postoperatively, all animals were executed in batch, with3animals out of each group, the integrated femurs were obtained and draped with sterile bandage after all soft tissues removed, saturated with saline, sealed with double layer plastic envelope, contained in refrigerator under-20℃circumstances. The specimens were thawed and contained in saline for2hours, then the maximum load and rigidity of the callus were measured using8874-Instron biomechanical testing machine. The size of the callus was measured and the bone mineral density surrounding the fracture site were measured using GE Prodigy double-energy X-ray bone densitometry machine before the biomechanical test. Three animals were executed on every observation time point in each group after1,2and3weeks postoperatively,1cm long specimen was abtained, centering on the fracture site, then divided into pieces about0.5cm×0.2cm×O.2cm using single plane razor blade, washed up with saline and put in4%paraformaldehyde fixed for72hours. Paraffin sections were made after decalcification and embedding. Histology of callus was observed after HE and others staining and expression and distribution of osteocalcin(oc) and collagen type Ⅱ(col2) were analyzed with immunohistochemical methods.Results:1. X-ray observations:After2weeks, the external callus increased obviously in Group two comparing with Group one, while the vagueness level of fracture line and bridging callus formation occurred later than the latter. The external callus increased obviously in Group three and Group four than that in the Group one, while the vagueness level of fracture line and bridging callus formation accelerated in the former groups.Statistically analysis was carried out after semiquantitative score was done according to the external callus formation and the vagueness level of fracture line. Two weeks after experiment was done, the score of the external callus formation and the vagueness level of fracture line were increased obviously in Group two and Group three than that in Group one and Group four(P<0.05). While three and five weeks after experiment, the score of the external callus formation and the vagueness level of fracture line were obviously lower in Group two than that in Group one(P<0.05), on the other hand, the scores were much higher in Group three and Group four than that in Group one (P<0.05)The time effect of controlled micromovement had various influence on the fracture healing of experimental animals. Although the external callus in Group two was more than that in the other Groups, but the vagueness of fracture line was obviously lower than other Groups. While in Group three and Group four, the external callus was larger than that in Group one but less than that in Group two. but the vagueness of fracture line was faster in Group three and Group four than that in Group one and Group two.2. Measurement of callus sizeFive weeks after experiment was done, the anteroposterior diameter and the exterior and interior diameter of the callus were obviously larger in Group two than that in Group one(P<0.05),but the diameters in Group three and Group four were smaller than that in Group one, although there was not statistically significant.3. Measurement result of bone mineral density.There was no significant difference of bone mineral density value and proportion rate in tne fracture site between Group two and Group one(P>0.05).But the value and proportion rate in tne fracture site were notably higher in Group there and Group four comparing with that in Group one (P<0.05)4. Biomechanical measurement results.The maximum load of the callus five weeks postoperatively was significantly higher in Group three and Group four than that in Group one and Group two(P<0.05), while the maximum load of the callus was notably lower in Group two than that in Group one (P<0.05). The deflection five weeks postoperatively was no difference between groups (P>0.05).The callus rigidity five weeks postoperatively was significantly higher in Group three than that in the other groups (P<0.05), while the callus rigidity was higher in Group four than that in Group one and Group two(P<0.05), but there was no difference between group one and two (P>0.05)5. Histology observation.One week postoperatively:organization hematoma between the fracture sides could be seen in all four experimental groups. Mass of cell aggregated together around the fracture ends to form fibrous callus, lots of inflammatory cells like mesenchyme cell、fibroblast、osteoblast、granulocyte、mononuclear macrophage and neonatal capillaries could be seen. Mesenchyme cells beyond the periosteum proliferated, differentiated to chondrocytes and osteoblasts, then to form neonatal bone trabecula. Osteoblasts proliferated actively in the margin of ossification zone under the periosteum, appearing1-2layers of osteoblasts. Chondrocytes began to appear, secreting cartilage matrix. And the neonatal chondrocytes in Group two were more than that in the other groups obviously. A small quantity of intramembrane osseous callus could be seen in all four experimental groups.Two weeks postoperatively:Partial cartilaginous callus connected between the fracture ends. Calcification front appeared in the juncture of chondrocytes and bone trabeculas formed under periosteum in all four groups. Endochondral ossification began to start, lots of osteoclasts occurred in Group three, osteoblasts activated all the more, appearing3-4layers of osteoblasts. Less neonatal bone trabeculas appeared in Group two, chondrocytes matured slower with fewer endochondral ossification. A few of woven bone presented.Three weeks postoperatively:similar to Group Four, osteoclasts presented in Group One, cartilage islands in the fibrous callus expanded gradually, majority of the chondrocytes surrounding blood vessel were spherical naive one with small volume. Chondrocytes arounding cartilaginous callus got to hypertrophy, and degenerated necrotic chondrocytes were substituted with primitive bone trabeculas. Lots of osteoclasts were still to remain in Group three, osteoblasts and endochondral ossification still activated, much woven periosteous callus extended to the fracture ends, with apparent blood vessel hyperplasia, partial callus crossed the fracture gap. While in Group two, chondrocytes matured slowly, endochondral ossification delayed, with sparse neonatal bone trabeculas and no callus crossing the fracture gap.6. Immunohistochemistry observation.Different degree increase of Osteocalcin and Collagen II were observed along with time prolonged in each group. One week postoperatively, The stain degree of Osteocalcin in group two was distinctly weaker than group three (P<0.05),while the Stain degree of Collagen II was distinctly stronger than other three groups (P<0.05); Two week postoperatively, The stain degree of Osteocalcin in group two was distinctly weaker than other three groups (P<0.05),while the stain degree of Collagen II in group one was distinctly weaker than group two and three (P<0.05):Three week postoperatively. The stain degree of Osteocalcin in group two was distinctly weaker than other three groups (P<0.05), and the stain degree of Osteocalcin in group three was distinctly stronger than group one (P<0.05),while the stain degree of Collagen Ⅱ in group one was distinctly weaker than other three groups (P<0.05)Conclusion:1. Occasion of micromovement is an important factor to influence the fracture healing, different time effect related to different onset of controlled micromovement. Micromovement early or late is unfavourable to fracture healing.2. Instant micromovement group gets inferior results to persistent fixation group, including healing time, bone mineral density in the fracture site and callus rigidity,although it has maximal and largest external callus.3. Micromovement begins on one and two weeks can significantly raise external callus formation and vagueness level of fracture line, accelerating bridging callus formation.4. Micromovement begins on one and two weeks can significantly raise bone mineral density and rigidity of callus.5. Micromovement begins on one and two weeks activated the osteoblasts even makes3-4layers of osteoblasts appeared,It also accelerates the maturity> hypertrophy and mineralization of chondrocyte,resulting in the stimulation of the fracture healing through endochondral ossification. Though the instant micromovement stimulate the formation of chondrocyte in the early stage,but slower the process of the maturity and hypertrophy and mineralization of chondrocyte6. Different degree increase of Osteocalcin and Collagen Ⅱ were observed along with time prolonged in each group.In instant micromovement group, The stain degree of Osteocalcin was distinctly weaker; Three week postoperatively, The stain degree of Osteocalcin in one week postoperative micromovement group was distinctly stronger than continuing immobilization group, while the stain degree of Collagen Ⅱ in continuing immobilization group was distinctly weaker than other three groups.7. Except that the micromovement begins on one and two weeks activated the osteoblasts, it seemingly can improve the amount and density of osteoclasts in callus to stimulate the maturity and mineralization of chondrocyte; The strengthening coupling of osteoblasts and osteoclasts can promote the transformation from soft callus to hard callus and the remolding of hard callus. |
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